KR20180039068A - Bone marrow-infiltrating lymphocyte (MIL) as a T-cell source for chimeric antigen receptor (CAR) - Google Patents

Abstract

In some embodiments, a marrow-infiltrating lymphocyte ("MIL") comprising a chimeric antigen receptor ("CAR") is provided. In some embodiments, embodiments provide a method of making a recombinant MIL, comprising: obtaining a bone marrow comprising an MIL; And transforming the MIL with a nucleic acid encoding a chimeric antigen receptor. In some embodiments, embodiments relate to a method of treating a condition in a subject, comprising administering to the subject an MIL comprising CAR.

Description

Bone marrow-infiltrating lymphocyte (MIL) as a T-cell source for chimeric antigen receptor (CAR)

Cross reference of related application

This application claims priority to U.S. Provisional Application No. 62 / 189,928, filed July 8, 2015, which is incorporated herein by reference in its entirety.

background

The majority of malignant tumor patients will die from their disease. One approach to treating these patients is to transgenic MILs to target antigens expressed on tumor cells through the expression of a chimeric antigen receptor ("CAR"). CAR is an antigen receptor designed to recognize cell surface antigens in a human leukocyte antigen-independent manner. In addition to the success of the CD19-targeted approach, attempts to use genetically modified cells expressing CAR to treat other malignancies have achieved limited success.

summary

In some embodiments, a marrow-infiltrating lymphocyte ("MIL") comprising a chimeric antigen receptor ("CAR") is provided. In some embodiments, the CAR comprises an extracellular domain capable of binding to a ligand. In some embodiments, the CAR comprises an intracellular domain that is capable of initiating an intracellular signaling cascade (e. G., In MIL).

In some embodiments, there is provided a method of treating a condition in a subject, comprising administering to the subject an MIL comprising CAR. In some embodiments, the method comprises administering to the subject a composition comprising an MIL population, wherein each MIL of the MIL population comprises CAR.

In some embodiments, there is provided a method of making a recombinant MIL, comprising: obtaining a bone marrow comprising an MIL; And transforming the MIL with a nucleic acid encoding a chimeric antigen receptor. Bone marrow can be obtained from the same subject as a subject with a neoplasm. The subject may be a human or a mouse.

details

In some embodiments, compositions and methods for treating cancer, including, but not limited to, hematologic malignancies and solid tumors are provided herein. Embodiments include, but are not limited to, the adoptive cell delivery strategy of bone marrow-infiltrating lymphocytes (MIL) transduced to express the chimeric antigen receptor (CAR). CAR is a molecule that produces a chimeric protein that exhibits specific anti-tumor cell immunoactivity by combining antibody-based specificity for a desired antigen (e.g., tumor antigen) with an MIL receptor-activated intracellular domain.

In some embodiments, the use of genetically modified MILs to stably express the desired CAR is provided. MIL expressing CAR is referred to herein as CAR-MIL or CAR-modified MIL. In some embodiments, the cell can be genetically modified to stably express the antibody binding domain on its surface, conferring new antigen specificity independent of MHC. In some embodiments, the MIL is genetically modified to stably express the CAR in which the antigen recognition domain of the specific antibody is combined with the intracellular domain of the CD3ζ chain or the Fc [gamma] RI protein into a single chimeric protein.

In some embodiments, the CAR comprises an extracellular domain having an antigen recognition domain, a transmembrane domain, and a cytoplasmic domain. In some embodiments, a transmembrane domain that is naturally associated with one of the domains in CAR is used. In some embodiments, the transmembrane domain can be selected by amino acid substitution to avoid binding of such domains to the transmembrane domain of the same or a different surface membrane protein to minimize interaction with other members of the receptor complex, . For example, the transmembrane domain may be a CD8 alpha hinge domain.

With respect to the cytoplasmic domain, for example, the CAR may be designed to combine with the CD28 and / or the 4-1BB signaling domain itself or in combination with any other desired cytoplasmic domain (s) useful in the environment of the CAR. In some embodiments, the cytoplasmic domain of CAR may be designed to further include the signaling domain of CD3ζ. For example, the cellular domain of CAR can include, but is not limited to, CD3ζ, 4-1BB and CD28 signaling modules, and combinations thereof. Thus, specific examples provide methods for their use for CAR-MIL and quantum therapy.

In some embodiments, the CAR-MIL can be generated by introducing into the cell a lentiviral vector comprising the desired CAR (e. G., CAR comprising the anti-CD19, transmembrane domain, and human 4-1BB) . CAR-MIL can, for example, be replicated in vivo, thus maintaining long-term persistence, leading to continued tumor control.

In some embodiments, the injection of CAR-MIL is provided to administer a gene-modified MIL that expresses CAR for the treatment of patients with neoplasia. In some embodiments, autologous infusion is used for treatment. The autologous MIL is collected from patients in need of treatment, activated and expanded using methods described herein and known in the art, and then injected back into the patient.

In some embodiments, an MIL expressing anti-CD19 CAR is used that includes both CD3ζ and 4-1BB co-stimulatory domains. In some cases, CAR MIL injected into a patient can remove leukemia cells in vivo. However, this embodiment is not limited to CD19 or MIL targeting the signal through the CD3ζ and / or 4-1BB mediated pathway. For example, embodiments include any antigen-binding domain that is fused with one or more intracellular domains selected from the group consisting of CD137 (4-1BB) signaling domain, CD28 signaling domain, CD3ζ signal domain, and any combination thereof Includes moieties.

Justice

The definitional articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical objects of the definite article. By way of example, "element" means one element or more than one element.

The terms "comprise," " comprise, "" comprise ", and " comprise ", as well as conjugates thereof as used herein, mean" including but not limited to. While various compositions and methods are described in the context of "comprising" various components or steps (as interpreted to mean "comprising"), the compositions, methods, Quot; comprise "or" comprise ", and such terms shall be interpreted as defining essentially a closed-member group.

"Activation" as used herein refers to the condition of the MIL stimulated sufficiently to induce detectable cell proliferation. Activation may also be associated with induced cytokine production, and detectable effector function. The term "activated MIL" refers specifically to the MIL undergoing cell division.

The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds an antigen. The antibody may be a full immunoglobulin derived from a natural or recombinant source, and may be an immunoreactive part of a full immunoglobulin. Antibodies can be present in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F (ab) 2, as well as single chain antibodies and humanized antibodies.

The term "antibody fragment" refers to a portion of an intact antibody and represents an antigenic determinant variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab ', F (ab') 2 and multispecific antibodies formed from Fv fragments, linear antibodies, scFv antibodies and antibody fragments.

The term "antigen" as used herein is defined as a molecule that causes an immune response. Such an immune response may include either or both of antibody production or activation of a specific immunologically-competent cell. Those skilled in the art will appreciate that virtually any macromolecule, including any protein or peptide, can act as an antigen. In addition, the antigen may be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that induces an immune response will thus encode an "antigen" like the term used herein. In addition, those skilled in the art will appreciate that the antigen need not be encoded alone by the full-length nucleotide sequence of the gene. Embodiments include, but are not limited to, the use of partial nucleotide sequences of more than one gene, and it is very apparent that these nucleotide sequences are arranged in various combinations to induce the desired immune response. In addition, those skilled in the art will understand that the antigen need not be encoded by a "gene" at all. It is very apparent that antigens can be produced as synthesized or can be derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

The term " anti-tumor effect ", as used herein, refers to a biological effect that may be manifested by a reduction in tumor volume, a reduction in tumor cell numbers, a reduction in metastasis, an increased expected life span, Quot; An "anti-tumor effect" may also be indicated by the ability of peptides, polynucleotides, cells and antibodies to prevent tumorigenesis first.

The term "self-antigen" means any self-antigen that is heterologous and is misidentified by the immune system. Self-antigens include, but are not limited to, cell proteins, phosphorous proteins, cell surface proteins, cell lipids, nucleic acids, and glycoproteins including cell surface receptors.

The term "autoimmune disease" as used herein is defined as a disorder resulting from an autoimmune reaction. Autoimmune diseases are the result of inadequate and excessive responses to self-antigens. Examples of autoimmune diseases include, but are not limited to, addison's disease, alopecia greata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guilin-Barre syndrome (Eg, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus psoriasis, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, and the like.

The term "autologous " as used herein refers to any material derived from the same entity to be re-introduced into the same individual afterwards.

"Allogeneic" refers to implants derived from different animals of the same species.

"Xenogeneic" refers to an implant derived from an animal of a different species.

The term "cancer" as used herein is defined as a disease characterized by rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast, prostate, ovarian, cervical, skin, pancreatic, colon, kidney, liver, brain,

As used herein, the term " co-stimulatory ligand "refers to a molecule on an antigen presenting cell (e.g., aAPC, dendritic cells, B cells, etc.) that specifically binds to a cognate co- And mediates MIL responses including, but not limited to, proliferation, activation, differentiation, and so forth, in addition to the primary signal provided by, for example, binding of a TCR / CD3 complex to a peptide loaded MHC molecule. Co-stimulatory ligands include but are not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Agents or antibodies that bind to the intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, , And a ligand that specifically binds to B7-H3. Also, the co-stimulatory ligand may be selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA- , NKG2C, B7-H3, and a ligand that specifically binds to CD83.

"Co-stimulatory molecule" refers to a cognate binding partner on the MIL that specifically binds to a co-stimulatory ligand and mediates a co-stimulatory response, such as, but not limited to, proliferation by MIL. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors.

As used herein, a " co-stimulatory signal "refers to a signal that, in combination with a primary signal such as TCR / CD3 ligand, leads to MIL proliferation and / or upregulation or down regulation of the major molecule.

"Disease" is a state of health of a subject in which the animal is unable to maintain homeostasis and the animal's health continues to deteriorate unless the disease improves. On the other hand, a "disorder" of a subject is a health condition that is less healthy than when the subject is able to maintain homeostasis but the subject's health condition is not impaired. If left untreated, the disorder does not necessarily result in an additional deterioration of the subject's health status.

An "effective amount" as used herein means an amount which provides a therapeutic or prophylactic benefit.

"Encoding" encompasses the use of specific nucleotide sequences, e. G., Gene (s), in a polynucleotide acting as a template for the synthesis of other polymers and macromolecules in the biological processes with defined nucleotide sequences (i.e., rRNA, tRAN and mRNA) , cDNA, or mRNA and the biological properties resulting therefrom. Thus, if the transcription and translation of the mRNA corresponding to the gene produces a protein in a cell or other biological system, the gene encodes the protein. Both the coding strand, i.e., the nucleotide sequence, which is identical to the mRNA sequence and commonly provided in the sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA, encodes a protein or other product of the gene or cDNA .

As used herein, "endogenous" refers to any material produced from or within an organism, cell, tissue or system.

The term "exogenous " as used herein refers to any material that is introduced into or out of an organism, cell, tissue, or system.

The term "expression" as used herein is defined as transcription and / or translation driven by its promoter of a particular nucleotide sequence.

"Expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; Other factors for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors can be any of those known in the art such as, for example, cosmids incorporating recombinant polynucleotides, plasmids (e. G., Contained in naked or liposomes) and viruses (e. G., Lentivirus, retrovirus , Adenovirus and adeno-associated virus).

"Homology" refers to sequence similarity or sequence similarity between two polypeptides or two nucleic acid molecules. If the positions in both the compared sequences are occupied by the same base or amino acid monomer subunit, for example, if the adenine is at a position in each of the two DNA molecules, then the molecule is homologous at that position. The percent homology between two sequences is a function of the number of matches of two sequences divided by the number of positions compared or the number of shared homology positions x 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparisons are made when two sequences are aligned to provide maximum homology.

The term "immunoglobulin" or "Ig" as used herein is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are often referred to as BCR (B cell receptor) or antigen receptors. The five members involved in this class of proteins are IgA, IgG, IgM, IgD, and IgE.

"Separated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated ", but the same nucleic acid or peptide that is partially or completely separated from its natural state coexistent material is" isolated ". The isolated nucleic acid or protein may be present in a substantially purified form, or may be present in a non-natural environment, such as, for example, a host cell.

The following abbreviations for commonly occurring nucleic acid bases, as used herein, are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

As used herein, "lentivirus" refers to the genus of retroviruses. Among the retroviruses, there is only one lentivirus that can infect non-dividing cells; Can transfer a significant amount of gene information into the DNA of host cells, which is one of the most efficient methods of gene transfer vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means of achieving a significant level of gene transfer in vivo.

The term "marrow infiltrating lymphocyte" ("MIL") as used herein refers to a lymphocyte derived from bone marrow. The marrow infiltrating lymphocyte ("MIL") has many distinguishable differences from peripheral blood lymphocytes as well as tumor infiltrating lymphocytes ("TIL"). The bone marrow ("BM") microenvironment is a specific immunological locus due to the abundance of antigen presenting cells ("APCs"). The presence of these antigen presenting cells maintains a higher level of central memory cells found in the bone marrow compartment for treatment and presentation of antigens. (Li JM et al J Immunol 2009 Dec 15; 183 (12): 7799-809). These MILs express memory markers such as CD45RO + and CD62L +, and there are more memory MILs than memory cells found in PBLs. (Noonan K et al Clin Cancer Res. 2012 Mar 1; 18 (5): 1426-34). In addition, MIL is not the only "TIL" of hematologic malignancy because of its ability to constantly prime memory cells to antigen (Beckhove et al., Clin. Invest. 2004 Jul 1; 114 (1) 76, Castiglioni P et al 6 J Immunol 2008; 180: 4956-4964). MIL also expresses more CXCR4 than their PBL counterparts due to the cognate antigenic epileptogenic factor type 1 ("SDF1"), which is expressed in large amounts in bone marrow stromal cells (Noonan K et al, Cancer Res. 2005 Mar 1; 65 (5): 2026-34). In addition, the expression of 41BB is probably increased in MIL compared to PBL due to the hypoxic nature of the BM micro-environment. In addition, MIL can be harvested and expanded in all patients as opposed to TIL (Noonan, K. et al. Sci. Transl. Med. 2015 May 20; 7 (288): 288ra78). TIL is found in only about 50% of patients, and only about 25% of patients contain expandable TIL. In contrast to peripheral blood lymphocytes (PBLs), MIL possesses a broad endogenous antigen repertoire that describes their inherent tumor specificity, a feature that is completely absent in PBL (Noonan et al Clin Cancer Res).

Unless otherwise specified, the term "nucleotide sequence encoding amino acid sequence" is the degraded version of each other and includes all nucleotide sequences encoding the same amino acid sequence. The nucleotide sequence encoding the protein and RNA may comprise an intron.

The term "operably linked" refers to the functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, leading to the expression of a heterologous nucleic acid sequence. For example, when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, link two protein coding regions in the same reading frame.

The term "over-expressing " the term" over-expressing "tumor antigen or tumor antigen refers to the expression of a tumor in a cell from a disease area, such as a solid tumor within the tissue or organ of a patient, It is intended to indicate the abnormal expression level of the antigen. Patients with solid tumors or hematologic malignancies characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

"Parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

The terms "patient," "subject," "subject," and the like are used interchangeably herein and refer to any animal or cell thereof that can be treated in the methods described herein, either in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

The terms "peptide "," polypeptide ", and "protein ", as used herein, are used interchangeably and refer to compounds comprising amino acid residues covalently joined by peptide bonds. A protein or peptide contains at least two amino acids, and there is no restriction on the maximum number of amino acids that can occupy a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked together by peptide bonds. As used herein, the term includes both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, and both long chains commonly referred to in the art as many types of present proteins . "Polypeptide" specifically includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

The term "promoter " as used herein is defined as a DNA sequence that is recognized by the synthetic machinery of the cell or the introduced synthetic machinery necessary to initiate the specific transcription of the polynucleotide sequence.

The term "promoter / regulatory sequence " as used herein refers to a nucleic acid sequence required for the expression of a gene product operably linked to a promoter / regulatory sequence. In some cases, such sequences may be core promoter sequences, and in other cases, such sequences may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may be, for example, a sequence that expresses the gene product in a tissue-specific manner.

A "persistent" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or characterizes a gene product, causes the gene product to be produced in the cell under most or all of the physiological conditions of the cell.

An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or characterizes a gene product, causes the gene product to be substantially produced in the cell only when a promoter corresponding to the promoter is present in the cell.

A "tissue-specific" promoter, when operably linked to a polynucleotide encoded or characterized by a gene, is a nucleotide sequence that causes the gene product to be substantially produced in the cell only when the cell is a cell of the tissue type corresponding to the promoter to be.

The term "stimulation" refers to the interaction of a stimulating molecule (e.g., a TCR / CD3 complex) with its cognate ligand to elicit a primary response induced by signal transduction events, such as, but not limited to, mediating signal transduction through a TCR / CD3 complex it means. Stimulation may mediate altered expression of certain molecules such as down-regulation of TGF-ss and / or reconstitution of the cytoskeletal structure and the like.

The term "stimulating molecule" as used herein refers to a molecule on the MIL that specifically binds to a cognate stimulating ligand present on an antigen presenting cell.

As used herein, a "stimulating ligand" refers to a cognate binding partner (referred to herein as a " stimulating molecule ") on an MIL when present on an antigen presenting cell (e.g., aAPC, dendritic cells, B cells, &Quot; means a ligand that specifically binds and mediates a primary response by MIL, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulating ligands are well known in the art and include in particular MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagent anti-CD28 antibodies, and superagent anti-CD2 antibodies.

The term "subject" is intended to include living organisms (e. G., Mammals) in which an immune response can be induced. Examples of subjects include humans, dogs, cats, mice, rats and their transgenic species.

The term "therapeutically" as used herein means treatment and / or prophylaxis. The therapeutic effect is obtained by inhibiting, alleviating or eradicating the disease state.

The term "therapeutically effective amount" refers to the amount of a subject compound that will elicit the biological or medical response of a tissue, system or subject sought by a researcher, veterinarian, physician or other clinician. The term "therapeutically effective amount" encompasses, in administration, an amount of a compound sufficient to prevent or to some extent alleviate the occurrence of one or more of the symptoms or symptoms of the disorder or disorder being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity, the age, weight, etc. of the subject being treated.

As used herein, "treating" a disease means reducing the frequency or severity of at least one symptom or symptom of the disease or disorder experienced by the subject.

The term " transfected "or" transformed "or" transduced, " as used herein, refers to the process by which an exogenous nucleic acid is delivered or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that is transfected with, transformed, or transfected with an exogenous nucleic acid. Cells include primary target cells and their progeny.

As used herein, the phrase "under transcriptional control" or "operably linked" refers to the presence of the promoter in the correct orientation and orientation with respect to the polynucleotide to control transcription initiation and expression of the polynucleotide by the RNA polymerase .

A "vector" is a composition of matter that includes isolated nucleic acids and that can be used to deliver isolated nucleic acids into cells. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphipathic compounds, plasmids, and viruses. Thus, the term "vector" includes plasmids or viruses that autonomously replicate. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate delivery of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

Explanation

The present disclosure provides compositions and methods for treating cancer among other diseases. The cancer may be a hematologic malignancy, a solid tumor, a primary or metastatic tumor. The cancer may be a hematologic malignancy such as chronic lymphocytic leukemia ("CLL"). Other diseases treatable using the compositions and methods described and provided herein include autoimmune diseases as well as viral, bacterial, and parasitic infections.

In some embodiments, cells engineered to express CAR (i. E., MIL), wherein CAR-MIL exhibits anti-tumor properties are provided. For example, CAR may be engineered to include an extracellular domain having an antigen-binding domain fused to the intracellular signaling domain of the MIL antigen receptor complex < RTI ID = 0.0 > zeta < / RTI > chain (e. For example, CAR, when expressed in MIL, can convert antigen recognition based on antigen-binding specificity. In some embodiments, the antigen is CD19, since such an antigen is expressed on malignant tumor B cells. However, embodiments are not limited to targeting CD19. Rather, this embodiment includes any antigen-binding moiety, wherein when such an antigen-binding moiety binds to its cognate antigen, it affects the tumor cells such that the tumor cells do not grow, Otherwise, the patient's tumor burden will be reduced or eliminated. The antigen-binding moiety may be fused with the intracellular domain from one or more of the co-stimulatory molecule and the [alpha] chain. In some embodiments, the antigen-binding moiety is fused with one or more intracellular domains selected from the group of the CD137 (4-1BB) signaling domain, the CD28 signaling domain, the CD3ζ signaling domain, and any combination thereof. The antigen-binding moiety may also be fused with an intracellular domain such as CD134 (OX40).

In some embodiments, the CAR comprises the CD137 (4-1BB) signaling domain. Without being bound by any particular theory, this is because embodiments are based in part on the discovery that CAR-mediated T cell responses can be further enhanced by the addition of a co-stimulatory domain.

I. Chimeric antigen receptor

A chimeric antigen receptor (CAR) comprising extracellular and intracellular domains is provided herein. The extracellular domain includes a target-specific binding element, otherwise referred to as an antigen-binding moiety. The intracellular domain, or alternatively the cytoplasmic domain, may comprise a part of the co-stimulatory signal transduction domain and / or the? Chain. The co-stimulatory signaling domain represents a portion of CAR that contains the intracellular domain of the co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigenic receptors or their ligands necessary for the efficient reaction of lymphocytes to antigens.

The spacer domain can be incorporated between the transmembrane and extracellular domains of CAR or between the transmembrane and cytoplasmic domains of CAR. The term "spacer domain" as used herein generally refers to a stretch of amino acids that serves to link the transmembrane domain to either the cytoplasmic domain or the extracellular domain in the polypeptide chain. The spacer domain may comprise up to 300 amino acids, preferably 2 to 100 amino acids, such as 25 to 50 amino acids.

II. Extracellular domain

In some embodiments, the CAR comprises a target-specific binding member, otherwise referred to as an antigen-binding moiety. The choice of moiety depends on the type and number of ligands that define the surface of the target cell. For example, an antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on a target cell associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands for antigen-binding domains in CAR include those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells. For example, the ligand may be a protein of bacteria, viruses or parasites. Similarly, the ligand may be an upregulated protein on the surface of cancer cells.

In some embodiments, the CAR can be engineered to engineer the desired antigen-binding moiety, which specifically binds to an antigen on the tumor cell, to target the tumor antigen of interest. As used herein, a "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with an hyperproliferative disorder" refers to a common antigen for a specific hyperproliferative disorder such as cancer. The antigens discussed herein are included by way of example only. The above list is not exclusive, and additional examples will be readily apparent to those skilled in the art.

Tumor antigens are proteins produced by tumor cells that cause an immune response, particularly a T-cell mediated immune response. The choice of antigen-binding moiety will depend on the particular type of cancer being treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, cancer embryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin- , RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), long chain carboxylase, mutant hsp70-2, M-CSF, prostase, prostate- (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostaglandin, PSMA, Her2 / neu, survivin, telomerase, prostate-carcinoma tumor antigen- ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF) -I, IGF-II, IGF-I receptor, and mesothelin.

In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express a number of proteins that can act as target antigens for immune attacks. Such molecules include, but are not limited to, prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in tumor-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostate cancer. Other target molecules belong to the family of transcription-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Another group of target antigens are tumor-fetal antigens, such as cancer embryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen unique to an individual tumor. B-cell differentiation antigens such as CD19, CD20 and CD37 are other candidate substances for target antigens of B-cell lymphoma. Some of these antigens (e. G., CEA, HER-2, CD19, CD20, idiotypes) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

The type of tumor antigen referred to may also be a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSA is unique to tumor cells and does not occur in other cells of the body. TAA-associated antigens are not native to tumor cells but are also expressed on normal cells under conditions that do not induce an immunological resistance state to the antigen. Expression of tumor antigens may occur under conditions that allow the immune system to respond to the antigen. TAAs can be antigens expressed on normal cells during fetal development when the immune system is immature and unresponsive, or they are present in extremely low levels in normal cells, but are expressed at much higher levels in tumor cells .

Non-limiting examples of TSA or TAA antigens include MART-1 / MelanA (MART-1), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2 and tumor- MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; Overexpressed embryo antigens such as CEA; Over-expressed cancer genes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; Unique tumor antigens originating from chromosomal translocations; For example, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; And viral antigens such as Epstein Barr virus antigen EBVA and human papilloma virus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, CA123, CD68, P1, CO-029, FGF-5, G250, Ga733, CA123, CA123, C-related protein, cyclophilin C-related protein, < RTI ID = 0.0 > TAAL6, TAG72, TLP, and TPS.

In some embodiments, the antigen-binding moiety portion of CAR is, but is not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3R ?, c- Met, PSMA, glycolipid F77, EGFRvIII, GD- ESO-1 TCR, MAGE A3 TCR, and the like.

Depending on the desired antigen to be targeted, the CAR may be engineered to include the appropriate antigen binding moiety specific to the desired antigen target. For example, if CD19 is the desired antigen to be targeted, the antibody to CD19 can be used as an antigen binding moiety for incorporation into the CAR. Thus, in some embodiments, the antigen-binding moiety portion of CAR targets CD19.

The extracellular domain of CAR may comprise, for example, a single-chain variable fragment ("scFv") linked to any of the targets described herein.

The extracellular domain may be any antigen-binding polypeptide, and a wide variety of such antigen-binding polypeptides are known in the art. In some cases, the antigen-binding domain is a single chain Fv ("scFv"). Other antibody-based recognition domains (cAb VHH (camel antibody variable domain) and humanized versions, IgNAR VH (shark antibody variable domain) and humanized versions, sdAb VH (single domain antibody variable domain) and "camelized" antibody variable domains In some cases, T-cell receptor (TCR) -based recognition domains, such as single chain TCRs (scTv, single chain 2-domain TCRs containing ννβ), are also suitable for use.

Other extracellular domains known in the art can also be used in embodiments (see, for example, PCT Patent Application Publication No. WO 2014/127261; U.S. Patent No. 8,975,071, incorporated herein by reference).

III. The transmembrane domain

With respect to the transmembrane domain, CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In some embodiments, a transmembrane domain that is naturally associated with one of the domains in CAR is used. In some cases, the transmembrane domain may be selected to avoid binding of such domains to the transmembrane domain of the same or a different surface membrane protein to minimize interaction with other members of the receptor complex, or may be modified by amino acid substitution .

The transmembrane domain can be derived from a natural source or the transmembrane domain can be designed (e.g., from a stretch of 18-30 hydrophobic amino acids such as alanine, valine, leucine and isoleucine to form a-helix). When the source is native, the domain may be derived from any membrane-bound or trans membrane protein. Specific uses of the transmembrane domain include the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154, Alternatively, a transmembrane domain may be designed, in which case it is primarily a hydrophobic moiety such as leucine (e.g., leucine, Phenylalanine, tryptophan, and / or tyrosine can be found in the vicinity of the membrane / water interface. Alternatively, short oligo- or polypeptides having from 2 to 10 amino acids in length can be found in the designed transmembrane domain. Linkers can link the cellular signaling and transmembrane domains of CAR. Glycine-serine spacers provide particularly suitable linkers.

IV. Intracellular domain

The cellular domain or otherwise the intracellular signaling domain of CAR is responsible for the activation of at least one of the normal effector functions of the MIL. The term "effector function" refers to a specific function of the cell. For example, the effector function of the MIL may be helper activity, including cytolytic activity or secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a portion of a protein that carries an effector function signal and directs the cell to perform a particular function. Whole intracellular signaling domains can be used, but in many cases the entire intracellular domain need not be used. To the extent that the truncated portion of the intracellular signaling domain is used, this truncated portion can be used instead of an intact chain as long as it carries an effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to deliver an effector function signal.

Some non-limiting examples of intracellular signaling domains for use in CAR include the cytoplasmic sequences of T-cell receptors (TCRs) and co-receptors that cooperate to initiate signal transduction after antigen receptor engagement, Any derivative or variant thereof, and any synthetic sequence having the same functional ability.

Signals generated through a single TCR are insufficient for complete activation of lymphocytes, and secondary or co-stimulatory signals are also required. Thus, MIL activation is mediated by two distinct classes of cytoplasmic signaling: a class that initiates antigen-dependent primary activation via TCR (primary cytosolic signaling sequence) and an antigen- A class that acts in an independent manner (secondary cytoplasmic signal transduction sequences).

Primary cytoplasmic signal transduction sequences regulate the primary activation of TCR complexes in a stimulatory or inhibitory manner. Primary cytoplasmic signal transduction sequences acting in a stimulatory manner may contain immunoreceptor tyrosine-based activation motifs or signaling motifs known as ITAM.

Examples of ITAM containing primary cytoplasmic signal transduction sequences that may be used include, but are not limited to, those derived from TCR ζ, FcR gamma, FcRbeta, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d . In some embodiments, the cytosolic signaling molecule of CAR comprises a cytoplasmic signaling sequence derived from CD3ζ.

In some embodiments, the cytoplasmic domain of CAR may be designed to include the CD3ζ signaling domain itself, or may be combined with any other desired cytoplasmic domain (s) useful in the environment of the CAR. For example, the cytoplasmic domain of CAR may comprise a portion of the CD3ζ chain and a co-stimulatory signaling region. The co-stimulatory signaling domain represents a portion of CAR that contains the intracellular domain of the co-stimulatory molecule. In some embodiments, the co-stimulatory molecule is a cell surface molecule other than an antigen receptor, or a ligand thereof that is required for efficient reaction by a lymphocyte to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7 -H3 and the like. Thus, while some embodiments may be illustrated as 4-1BB as a co-stimulating signaling element, other co-stimulating elements may also be used.

The cytoplasmic signaling sequences within the cytoplasmic signaling portion of CAR can be linked together in a random or specific sequence. Alternatively, preferably short oligo- or polypeptide linkers of 2 to 10 amino acids in length can form linkages. Glycine-serine spacers provide particularly suitable linkers.

In some embodiments, the cytoplasmic domain is designed to include the signaling domain of CD3ζ and the signaling domain of CD28. In some embodiments, the cytoplasmic domain is designed to include the signaling domain of CD3ζ and the signaling domain of 4-1BB. In some embodiments, the cytoplasmic domain is designed to include the signaling domain of CD3ζ and the signaling domain of CD28 and 4-1BB.

In some embodiments, the cytosolic domain in CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3ζ.

V. vector

Expression of a natural or synthetic nucleic acid encoding CAR is typically accomplished by operatively linking a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and introducing the construct into an expression vector. The vector may be suitable for replication and integrated eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for the regulation of the expression of the desired nucleic acid sequence.

Vectors derived from retroviruses such as lentivirus are suitable tools for achieving long-term gene transfer since they permit long-term stable integration of transgene genes and their proliferation in daughter cells. Lentiviral vectors have additional advantages in that they can transduce non-proliferating cells, such as hepatocytes, as compared to vectors derived from tumor-retroviruses such as leukemia and animal leukemia virus. They also have the additional advantage of low immunogenicity.

Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, for example, by screening a library from cells expressing the gene or from a vector containing the gene, ≪ RTI ID = 0.0 > techniques, < / RTI > Alternatively, the gene of interest may be generated by synthesis rather than cloned.

Expression constructs can also be used for nucleic acid immunization and gene therapy using standard gene transfer protocols. Methods for gene delivery are known in the art (see, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, incorporated herein by reference). In some embodiments, embodiments provide a gene therapy vector. The nucleic acid sequence may also be inserted, without limitation, using gene editing techniques such as CRISPR.

Nucleic acid can be cloned into several kinds of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and cosmids. A particular interest vector includes an expression vector, a replication vector, a probe generation vector, and a sequencing vector.

The expression vector may be provided to the cell in the form of a viral vector. Viral vector techniques are well known in the art and are described, for example, in Green & Sambrook ( Molecular Cloning: A Laboratory Manual, (4th ed., 2012)) and other virus and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses. In general, suitable vectors contain functional replication origin, promoter sequences, convenient restriction endonuclease sites, and one or more selectable markers in at least one organism (e.g., WO 01/96584; WO 01/29058; And US Patent No. 6,326,193).

Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene may be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of the subject in vivo or ex vivo. In some embodiments, adenoviral vectors are used. In some embodiments, lentiviral vectors are used.

Additional regulatory elements (e. G., Promoters and enhancers) regulate the frequency of transcription initiation. Typically, they are located in the 30-100,000 bp upstream of the starting site, but a large number of promoters have recently also been shown to contain functional elements downstream of the starting site. The spacing between promoter elements is often flexible, and the promoter function is preserved when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activation reduction begins. Depending on the promoter, the individual elements may act cooperatively or independently to activate transcription.

One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. Such a promoter sequence is a strong allosteric promoter sequence capable of inducing high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the growth factor-1 alpha (EF-1 alpha). But are not limited to, the promoters of simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein- Virus immediate immediate early promoter, Rous sarcoma virus promoter, as well as other allosteric promoter sequences including human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter and the creatine kinase promoter. The promoter is not limited to the constant promoter. Inducible promoters may also be used. The use of an inducible promoter allows for a molecular switch that can turn on the expression of the operably linked polynucleotide sequence when such expression is required or turn off expression when expression is not required to provide. Examples of inducible promoters include, but are not limited to, metallothionine promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or reporter gene, or both, so that the expression vector from the cell population to be transfected or infected through the viral vector Can be easily identified and selected. In other embodiments, selectable markers may be carried on separate DNA fragments and used in co-transfection procedures. Both selectable marker and reporter genes can be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic-resistant genes such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to assess the function of regulatory sequences. Generally, the reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and that encodes a polypeptide exhibiting some characteristics that are easily detectable by expression, for example, enzyme activity. Expression of the reporter gene is assayed at the appropriate time after the DNA is introduced into the recipient cell. Suitable reporter genes may include luciferase, beta-galactosidase, chloramphenicol acetyltransferase, a gene encoding secreted alkaline phosphatase, or a green fluorescent protein gene (see, for example, Ui-Tei et al., 2000 FEBS Letters 479: 79-82). In some embodiments, the reporter gene is mCherry.

Methods for introducing and expressing genes into cells are well known in the art. With respect to expression vectors, the vector may be readily introduced into host cells, e. G., Mammalian, bacterial, yeast or insect cells by any method known in the art. For example, an expression vector can be delivered into a host cell by physical, chemical, or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle impact, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art (see, for example, Green & Sambrook , Molecular Cloning: A Laboratory Manual , (4th ed., 2012) .

Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors and, in particular, retroviral vectors have become widely used methods for inserting genes into mammalian cells. Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, adeno-associated virus, and the like (see, for example, U.S. Patent Nos. 5,350,674 and 5,585,362).

Chemical means for introducing the nucleic acid into the host cell include lipid-based systems including colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and oil-in-water emulsions, micelles, mixed micelles, and liposomes . An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e. G., An artificial vesicle).

When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introducing the nucleic acid (in vitro, in vitro or in vivo) into a host cell. In another embodiment, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid can be encapsulated within the aqueous interior of the liposome or can be attached to the liposome via a linking molecule scattered within the lipid bilayer of the liposome or associated with both the liposome and the oligonucleotide, Or may be dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contain micelles, form complexes with micelles, or otherwise associate with lipids have. A lipid, lipid / DNA, or composition associated with a lipid / expression vector is not limited to any particular structure in solution. For example, they may exist as a micelle in a bilayer or "collapsed" structure. They may also simply disperse in solution and possibly form aggregates of uneven size or shape.

VI. Bone marrow infiltrating lymphocyte

Prior to MIL expansion and gene modification, sources of MIL are obtained from the subject. In patients with any number of types of cancer, including hematological malignancies and solid tumors, T cells can be readily obtained from a myelocytic microenvironment with increased tumor specificity compared to peripheral blood (see, for example, U.S. Patent Application Publication No. US 2011/0223146, which is incorporated herein by reference). By comparing T cells obtained from these two different compartments from subjects with hematologic malignancies, an oligoclonal restriction of bone marrow infiltrating lymphocytes (MIL) obtained from bone marrow aspiration is observed. Methods such as those involving anti-CD3 / CD28 antibody-conjugated magnetic beads can be used to activate and expand bone marrow cells in vitro to generate activated MIL. Activated MIL shows greater expansion and improved tumor activity compared to peripheral blood lymphocytes in all tested patients. These findings include 1) the bone marrow is a reservoir of tumor-specific T cells; 2) MIL can be activated and expanded in all studied patients (compared to the limited number observed in metastatic melanoma); 3) these cells migrate to the bone marrow upon injection; 4) lasts up to 200 days after adoptive transfer in NOD / SCID mice; 5) activated MIL suggests the inclusion of extensive antigen recognition as it can eradicate pre-established diseases and target myeloma stem cell precursors.

T-cells representing a minority of the population of total bone marrow cells can be expanded in the presence of nearly complete bone marrow. To ensure maximal tumor-T cell contact, the inhaled bone marrow can be fractionated with a density gradient of lymphocyte separation medium and cells can be collected up to nearly the level of the red blood cell pellet. This separation method substantially removes only red blood cells and neutrophils, providing nearly complete bone marrow, and inducing the collection of both T cells as well as tumor cells. T-cells can be expanded without a T-cell specific separation step and without a tumor cell isolation step. Cell type specific separation steps include, for example, classification using cell labeling and fluorescence activated cell sorting (FACS) using antibodies or other cell-type specific detectable labels. In some embodiments, the method can be practiced without such labeling and cell sorting methods.

For activation of the beads, bead-T cell contact is preferably maximized during the first 24-48 hours of incubation. Since T-cells represent only a small number of total cells in a population, contact between antibody coated beads and T-cells is about 1: 1 to about 5: 1 bead versus cell, preferably about 2: Is promoted using a sufficient number of beads in the cells to a range of about 4: 1 bead versus cell, more preferably about 2.5: 1 to 3.5: 1 bead versus cell. Such ratios are applicable to the described beads, and variations in the size of the beads and / or the antibody density on the beads may change the bead: cell ratio.

In some embodiments, the device can be used to culture cells, which provides a smooth, hard, round bottom surface to facilitate collection of cells and beads close by gravity (see, for example, U.S. Patent Application Public number US 2011/0223146). The apparatus comprises a closed cell vessel resting on a support. During at least the first three days of culture in the presence of beads, the container is preferably stationary (i.e., without locking or rotating) to further promote contact between the bead and the cell. These steps and conditions are desirable to maximize the expansion of the tumor-specific MIL using beads to enable the production of sufficient cells to be therapeutically useful. In addition, these culture conditions promote the growth of T cells without promoting the growth of tumor cells.

Several aspects of MIL make them candidates for immunotherapy. Specifically, under the conditions described herein, they expand more rapidly upon stimulation than PBL, often maintaining an asymmetric T-cell repertoire upon activation suggesting perhaps enhanced tumor specificity. While the inactivated MILs show a severe degradation response to autologous tumors, the ability to activate and expand T cells and significantly improve their tumor response is a presumed mechanism mediating T- Against tolerance. In addition, activated MILs show tumor specificity with little cross-reactivity to non-malignant tumor hematopoietic factors, with higher expression of CXCR-4, greater reactivity to SDF-I, Of the mobile phone. Collectively, this finding activates and extends bone marrow-infiltrating T cells with memory / effector phenotypes that appear to target a wide range of tumor antigens present in not only terminally differentiated mature plasma cells but also their precursors, Demonstrating the ability to have chemokine receptors that appear to promote trafficking - a feature that is necessary to maximize anti-tumor immunity of adoptive immunotherapy.

Activation and expansion of MIL is based on two previously reported phenomena: improved tumor specificity of tumor-infiltrating lymphocytes (Rosenberg et al. Science 1986; 233: 1318) and melanoma (Letsch et al. Cancer Res 2003; 63 : 5582-6), breast cancer (Feuerer et al., Nat Med 2001; 7: 452), and multiple myeloma-bone marrow also have a tumor microenvironment (Dhodapkar et al., Proc Natl Acad Sci USA 2002; 99: 13009) Gt; T-cell < / RTI >

The ability to activate and expand MIL as a means to significantly increase tumor specificity and overcome unresponsiveness compared to activated PBL is provided herein. The presence of the tumor in the marrow microenvironment may play an important role in preserving the antigen specificity of activated MIL. Some hypotheses can explain the increased reactivity of activated MILs over activated PBLs. Without being bound by a mechanism, this suggests that persistence of the antigen in the bone marrow may be essential for the maintenance of memory response. Anti-CD3 / CD28 antibody-coated bead activation may be reversible in the bone marrow T-cell population. Similarly, plate bound and / or soluble CD3 and / or CD28 can be used for activation. However, the means for activating the MIL is not particularly limited, and any suitable activation method may be used in various embodiments. As demonstrated herein, the tumor specificity of activated MIL was dependent on the presence of antigen during T-cell activation. In addition, bone marrow is a functional lymphatic organ capable of initiating both a primary immune response and a secondary response through reactive lymphoid follicles in the presence of risk signals (infection, inflammation, autoimmune and cancer).

In myeloma patients, T cells show significant asymmetry of the VB T-cell receptor repertoire. This asymmetry results from the selective proliferation of T cells with prominent tumor specificity or from the very underlying T-cell defect characteristics of patients with significant tumor burden. In the latter case, the advantage of polyclonal stimulation of PBL with anti-CD3 / CD28 antibody-conjugated magnetic beads is the ability to restore normal T-cell repertoire and thus correct any underlying T-cell defects. In contrast, when oligoclonal expression of a particular VB family reflects the presence of T cells with tumor specificity, activation and expansion of such T cell pools with retained antitumor activity and T-cell receptor repertoire asymmetry is desirable . As demonstrated herein, PBL normalized their VB T-cell repertoire upon activation and expansion of anti-CD3 / CD28 antibody-conjugated magnetic beads, while MIL maintained VB restriction. Given the enhanced tumor-specific response of activated MIL, these asymmetric T-cell repertoires may imply greater tumor recognition. Without being bound to a mechanism, it may be important to preserve and possibly increase the degree of VB asymmetry during T-cell expansion.

Activation and expansion of MILs in anti-CD3 / CD28 antibody-conjugated magnetic beads produce potent anti-tumor activity, and persistence of the antigen during such expansion can be critical to maintaining (and increasing) tumor specificity. Dhodapkar et al. (2002) also studied the role of MIL in patients with myeloma. Similar to our findings, the newly isolated MIL or PBL did not show activity upon stimulation with autologous tumor or tumor peptides. In this study, however, no significant difference was observed between peripheral blood and T cells obtained from the myeloid compartment in the enzyme-binding immuno-spot assay after 12-16 days incubation with tumor-pulsed dendritic cells, but 10 times An excess of activated MIL antitumor response was observed in our system in all assays tested. These inconsistent results may be related to the efficacy of anti-CD3 / CD28 bead stimulation compared to dendritic cell activation of MIL. Without being bound to the mechanism, what appears to be an increase in the frequency of tumor-reactive T cells in activated and expanded MIL may reflect functional recovery and resistance destruction of tumor-reactive T cells. In addition, the stimulation of MIL in the marrow microenvironment is another important factor that can explain these results.

Enrichment of the MIL population by negative selection can be achieved with a combination of antibodies directed to the cell-specific surface markers selected by the negative. One method is cell sorting and / or selection through negative autoimmune adherence or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the selected cells by negative. For example, to enrich CD4 + cells by negative selection, monoclonal antibody cocktails typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8.

For separation of the desired cell population by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume at which beads and cells are mixed together (i. E., Increase cell concentration) to ensure maximum contact of the beads with the cells.

In some embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting a mixture of MIL and surface (e.g., particles, such as beads), the interaction between particles and cells is minimized. It is selected for cells that express a large amount of the desired antigen to be bound to the particle.

Also provided herein are collections of samples comprising MIL from a subject for a predetermined period of time before extended cells as described herein may be needed. As such, the source of the cell to be expanded can be harvested at any point in time, and the desired cells, such as MIL, can be used for MIL therapy for any number of diseases or conditions that would benefit from MIL therapy as described herein It can be separated and frozen for later use. In some embodiments, a sample comprising an MIL is generally taken from a healthy subject. In some embodiments, the sample comprising MIL is taken from a generally healthy subject at risk of developing the disease but not yet disease, and the cell of interest is isolated and frozen for later use. In some embodiments, the MIL is expanded, frozen, and used later. In some embodiments, the sample is collected from a patient immediately after diagnosis of a particular disease as described herein, but before any treatment. In some embodiments, the cells include, but are not limited to, agents such as, for example, natalizumab, epalgipath, an antiviral agent, a chemotherapeutic agent, radiation, an immunosuppressant such as cyclosporine, azathioprine, methotrexate, mycophenolate, , Or other immunotherapeutic agents such as the CAMPATH, anti-CD3 antibody, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, Is separated from the sample containing the MIL from the subject before the form. These drugs inhibit calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (laminectin), which is important for growth factor-induced signaling. In some embodiments, the cells are isolated for the patient and used to treat bone marrow or stem cell transplantation, chemotherapeutic agents such as MIL ablation therapy using fludarabine, external-beam radiation therapy (XRT), cyclophosphate (For example, before, concurrently with, or after) an antibody, e. G. In some embodiments, the cells are first isolated and frozen for further use for treatment following B-cell resection therapy, such as agents that react with CD20, e.g., Rituxan.

In some embodiments, the MIL is obtained from the patient immediately after treatment. In this regard, the quality of the MIL obtained after treatment with the following specific cancer treatments, particularly after treatment with a drug that damages the immune system, during which the patient generally recovers from treatment, is optimized for the ability to expand in vitro Or improved . Likewise, after in vitro manipulation using the methods described herein, these cells may be in a desirable state for improved engraftment and in vivo expansion. Thus, the MIL can be collected during this recovery phase.

Regardless of whether the MIL is genetically modified to express the preferred CAR, the MIL is generally described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232, 566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; And US Patent Application Publication No. 20060121005 (which is incorporated herein by reference).

In some embodiments, the MIL is extended by contact with agents that stimulate CD3 / TCR complex-related signals and with ligand-attached surfaces that stimulate co-stimulatory molecules on the MIL surface. In particular, the MIL population may be produced by contacting an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, as described herein, or with a protein kinase C activator (e. G., Bryostatin). ≪ / RTI > In the co-stimulation of the accessory molecule on the surface of the MIL, a ligand that binds to the accessory molecule is used. For example, the MIL population may be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable to stimulate MIL proliferation. To stimulate the proliferation of CD4 + MIL or CD8 + MIL, anti-CD3 antibodies and anti-CD28 antibodies may be contacted. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which may be used as may be used in other methods generally known in the art (Berg et al , J. Immunol Meth. 227 (1- (2-amino-1-phenylpropionyl) -1,2,3,4-tetrahydronaphthalene- 2): 53-63, 1999).

In certain embodiments, the primary stimulus signal and the co-stimulus signal for the MIL may be provided in different protocols. For example, the agent that provides each signal may be in solution or coupled to a surface. When coupled to a surface, the agent can be coupled to the same surface (i.e., "cis" formation) or isolated surface (i.e., "trans" formulation). Alternatively, one agent may be coupled to the surface and the other agent may be in solution. In some embodiments, the agent that provides the co-stimulatory signal is bound to the cell surface and the agent that provides the primary activation signal is present in solution or coupled to the surface. In certain embodiments, both agents may be present in solution. In some embodiments, the agent may be in a soluble form and then cross-linked to a surface, such as a cell or antibody that expresses an Fc receptor or other binding agent to be bound to the agent. (See, among other things, U.S. Patent Application Publication Nos. 20040101519 and 20060034810, which are incorporated herein by reference in their entirety for an artificial antigen presenting cell (aAPC) considered for use in the activation and expansion of MIL).

In some embodiments, the two agents are immobilized on the bead with the same bead, i.e., "sheath " or separate bead, or" trans. " For example, the agent that provides the primary activation signal is an anti-CD3 antibody or antigen-binding fragment thereof, and the agent that provides the co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; Both agents are co-immobilized in the same bead at equivalent molecular weights. In some embodiments, a 1: 1 ratio of each antibody bound to beads is used for CD4 + MIL expansion and MIL growth. In some embodiments, the ratio of the anti-CD3: CD28 antibody bound to the bead is used so that the increase in MIL expansion is comparable to the observed extension using a ratio of 1: 1. In some embodiments, an increase of about 1-fold to about 3-fold is observed compared to the expansion observed using a ratio of 1: 1. In some embodiments, the ratio of CD3: CD28 antibody bound to beads is in the range of 100: 1 to 1: 100 and is any integer value in between. In some embodiments, more anti-CD28 antibody is bound to the particles than the anti-CD3 antibody, i. E. The ratio of CD3: CD28 is less than one. In some embodiments, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to beads is greater than 2: 1. In some embodiments, a 1: 100 CD3: CD28 ratio of the antibody bound to the bead is used. In some embodiments, a 1: 75 CD3: CD28 ratio of the antibody bound to the bead is used. In some embodiments, a 1: 50 CD3: CD28 ratio of the antibody bound to the bead is used. In some embodiments, a 1:30 CD3: CD28 ratio of the antibody bound to the bead is used. In some embodiments, a 1: 10 CD3: CD28 ratio of the antibody bound to the bead is used. In some embodiments, a 1: 3 CD3: CD28 ratio of the antibody bound to the bead is used. In some embodiments, a 3: 1 CD3: CD28 ratio of the antibody bound to the bead is used.

1: 500 to 500: 1, and the ratio of particle to cell of any integer between them can be used to stimulate the MIL. As one skilled in the art will readily appreciate, the ratio of particle to cell can depend on the particle size versus the target cell. For example, smaller size beads can be bound to only a few cells, while larger beads can be much more. In certain embodiments, the ratio of cell to particle is in the range of 1: 100 to 100: 1 and any integer value therebetween; in a further embodiment, the ratio comprises 1: 9 to 9: 1, Can also be used to stimulate the MIL. The percentage of cells that result in anti-CD3- and anti-CD28-coupled particles versus MIL stimulation may vary as described above, but specific values include, but are not limited to, 1: 100, 1:50, 1:40 1: 1, 1: 2, 1: 2, 1: 2, 1: : 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1 and 15: 1. In some embodiments, the ratio is at least 1: 1 particles per cell. In some embodiments, a ratio of particle to cell of 1: 1 or less is used. In some embodiments, the particle: cell ratio is 1: 5. In some embodiments, the ratio of particle to cell can vary depending on the stimulation day. For example, in some embodiments, the ratio of particle to cell is 1: 1 to 10: 1 on the first day, and then the additional particles are dispersed in a final ratio of 1: 1 to 1:10, (Based on the number of cells on the day of addition). In some embodiments, the ratio of particle to cell is 1: 1 on the first day of stimulation and 1: 5 on the third day and fifth day of stimulation. In some embodiments, the particles are added daily or every other day in a final ratio of 1: 1 on the first day of stimulation and 1: 5 on days 3 and 5 of stimulation. In some embodiments, the ratio of particle to cell is 2: 1 on the first day of stimulation and 1:10 on the third day and fifth day of stimulation. In some embodiments, the particles are added daily or every other day in a final ratio of 1: 1 on the first day of stimulation and 3:10 on day 3 and 5:10. It will be understood by those skilled in the art that various other ratios may also be used. For example, the ratio will depend on the particle size, cell size and type.

In some embodiments, the MIL is combined with a formulation-coated bead, followed by separation of the beads and cells, followed by incubation of the cells. In some embodiments, prior to incubation, the formulation-coated beads and cells are not separated, but are co-cultured. In some embodiments, the beads and cells are first enriched by applying a force, e.g., magnetic force, to induce increased ligation of cell surface markers, thereby inducing cellular stimulation.

For example, cell surface proteins can be ligated by allowing paramagnetic beads (3 x 28 beads) to attach to anti-CD3 and anti-CD28 to contact the MIL. In some embodiments, cells and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads in a 1: 1 ratio) are combined in a buffer, preferably PBS (without divalent cations such as calcium and magnesium) do. One skilled in the art will readily recognize that any cell concentration can be used. For example, the target cell can be very rare in the sample and can only account for 0.01% of the sample, or the entire sample (i.e., 100%) can occupy the target cell of interest. Thus, any number of cells can be used. In certain embodiments, it may be desirable to significantly reduce the volume at which particles and cells are mixed together (i. E., Increase cell concentration) to ensure maximum contact between the cells and the particles. For example, in some embodiments, a concentration of about 2 billion cells / ml is used. In some embodiments, more than 100 million cells / ml are used. In some embodiments, cell concentrations of 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 50 million cells / ml are used. In some embodiments, cell concentrations of 7500, 8000, 8500, 9000, 95 million or 100 million cells / ml are used. In some embodiments, concentrations of 125 million or 150 million cells / ml may be used. The use of high concentrations can lead to increased cell yield, cell activation and cell expansion. In addition, the use of high-density cells allows a more efficient capture of cells capable of weakly expressing the target antigen of interest, such as CD28-negative cells. Such cell populations may have therapeutic value and may be desirable in certain embodiments.

In some embodiments, the mixture can be incubated for a number of hours (about 3 hours) to about 14 days, or any integer value times in between. In some embodiments, the mixture can be cultured for 21 days. In some embodiments, the beads and MIL are incubated together for about 8 days. In some embodiments, the beads and MIL are incubated together for about 2-3 days. Several stimulation cycles may also be desired so that the incubation time of the MIL can be 60 days or more. Conditions suitable for MIL culturing include serum (e.g., fetal or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM- (E.g., at least essential, which may contain factors necessary for proliferation and survival, including IL-12, IL-15, TGFβ and TNF-α or any other additive for cell growth known to those skilled in the art Media (Minimal Essential Media) or RPMI Media 1640 or X-vivo 15 (Lonza). Other additives for cell growth include, but are not limited to, surfactants, plasmenates, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium may include RPMI 1640, AIM-V, DMEM, MEM, alpha-MEM, F-12, X-Vivo 15 and X-Vivo 20, (S) sufficient for growth and expansion of serum-free or serum (or plasma) or a defined set of hormones and / or MILs. Antibiotics, for example, penicillin and streptomycin are included only in the experimental culture and not in the cell culture to be injected into the subject. Target cells are the conditions required to support growth, for example, is kept under appropriate temperature (e.g., 37 ℃) and atmosphere (e.g., air + 5% CO ₂₎ condition.

In addition to the CD4 and CD8 markers, other phenotypic markers are significantly different, but mostly reproducible during the course of the cell expansion process. Thus, this reproducibility enables the ability to customize activated MIL products for specific purposes.

In addition, a method for producing tumor invading lymphocytes can be used to make MILs. For example, high-dose IL-2 growth conditions can be used to generate "young" TILs and these methods are applicable to the manufacture of MILs (see, for example, U.S. Patent No. 8,383,099 Reference).

In some embodiments, the MIL may also be activated and / or expanded under hypoxic conditions. An example of growing MIL under hypoxic conditions can be found, for example, in WO2016037054, which is incorporated herein by reference in its entirety.

In some embodiments, the method comprises: removing cells from bone marrow, lymphocytes and / or marrow infiltrating lymphocytes ("MIL") from a subject; Incubating the cells in a hypoxic environment to generate an activated MIL; Lt; RTI ID = 0.0 > MIL < / RTI > Cells can also be activated in the presence of anti-CD3 / anti-CD28 antibodies and cytokines as described herein. Cytokines may also be used to activate MILs as described herein. Nucleic acid molecules encoding CAR, such as one of those described herein, may be transfected or infected into a cell either before or after the MIL is incubated in a hypoxic environment.

The hypoxic environment includes less than about 21% oxygen such as about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10% , 7%, 6%, 5%, less than 4%, or less than about 3% oxygen. For example, a hypoxic environment can include from about 0% oxygen to about 20% oxygen, such as from about 0% oxygen to about 19% oxygen, from about 0% oxygen to about 18% oxygen, from about 0% From about 0% oxygen to about 13% oxygen, from about 0% oxygen to about 12% oxygen, from about 0% oxygen to about 16% oxygen, from about 0% From about 0% oxygen to about 8% oxygen, from about 0% oxygen to about 7% oxygen, from about 0% oxygen to about 11% oxygen, from about 0% oxygen to about 10% From about 0% oxygen to about 6% oxygen, from about 0% oxygen to about 5% oxygen, from about 0% oxygen to about 4% oxygen, or from about 0% oxygen to about 3% oxygen. In some embodiments, the hypoxic environment comprises about 1% to about 7% oxygen. In some embodiments, the hypoxic environment comprises from about 1% to about 2% oxygen. In some embodiments, the hypoxic environment comprises from about 0.5% to about 1.5% oxygen. In some embodiments, the hypoxic environment comprises from about 0.5% to about 2% oxygen. The hypoxic environment is approximately 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7% %, 4%, 3%, 2%, 1% or about 0% oxygen. In some embodiments, the hypoxic environment comprises about 7%, 6%, 5%, 4%, 3%, 2% or 1% oxygen.

In a hypoxic environment, the MIL incubation is performed for at least about 1 hour, such as at least about 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 60 hours, 3 days, 4 days, And incubating the MIL in a tissue culture medium, e.g., for 1, 7, 8, 9, 10, 11, 12, 13 or even at least about 14 days. Incubation can include incubating the MIL for from about 1 hour to about 30 days, such as from about 1 day to about 20 days, from about 1 day to about 14 days, or from about 1 day to about 12 days. In some embodiments, the MIL incubation in a hypoxic environment comprises incubating the MIL in a hypoxic environment for about 2 to about 5 days. The method comprises administering an MIL in a hypoxic environment for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, ≪ / RTI > In some embodiments, the method comprises incubating the MIL in a hypoxic environment for about 3 days. In some embodiments, the method comprises incubating the MIL in a hypoxic environment for about 2 days to about 4 days. In some embodiments, the method comprises incubating the MIL in a hypoxic environment for about 3 to about 4 days.

In some embodiments, the method further comprises incubating the MIL in a hypoxic environment, for example, and then incubating the MIL in a normal oxygen environment.

The normal oxygen environment may include at least about 21% oxygen. The normal oxygen environment may include from about 5% oxygen to about 30% oxygen, such as from about 10% oxygen to about 30% oxygen, from about 15% oxygen to about 25% oxygen, from about 18% oxygen to about 24% About 23% oxygen, or about 20% oxygen to about 22% oxygen. In some embodiments, the normal oxygen environment comprises about 21% oxygen.

In a normal oxygen environment, the MIL incubation is performed for at least about 1 hour, such as at least about 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 60 hours, 3 days, 4 days, 6, 7, 8, 9, 10, 11, 12, 13 or even at least about 14 days in a tissue culture medium. Incubation can include incubating the MIL for about 1 hour to about 30 days, such as about 1 day to about 20 days, about 1 day to about 14 days, about 1 day to about 12 days, or about 2 days to about 12 days .

In some embodiments, the cell is transfected or infected with a nucleic acid molecule encoding a CAR described herein after being placed in a normal oxygen environment or in a normal oxygen environment.

In some embodiments, the MIL is obtained by extracting a bone marrow sample from a subject and culturing / incubating the cells as described herein. In some embodiments, bone marrow samples are centrifuged to remove erythrocytes. In some embodiments, the bone marrow sample is not treated with, or obtained by, component harvesting. In some embodiments, the bone marrow sample does not contain peripheral blood lymphocytes ("PBL") or the bone marrow sample contains substantially no PBL. This method is selected for cells that are not identical to those known as TIL. Thus, MIL is not a TIL.

In some embodiments, the cells can be plated in plates, flasks, or bags. In some embodiments, the hypoxic condition can be achieved by flushing the hypoxic chamber or cell culture bag with a 95% nitrogen and 5% CO ₂ gas mixture for 3 minutes. This may cause, for example, 1-2% or less of the O ₂ gas in the receptacle. The cells can then be cultured as described herein or in the examples of WO2016037054, which is incorporated herein by reference.

In some embodiments, a hypoxic MIL is provided comprising CAR as described herein. In some embodiments, the hypoxic MIL is in the environment of about 0.5% to about 5% oxygen gas. In some embodiments, the hypoxic MIL is in the environment of about 1% to about 2% oxygen gas. In some embodiments, the hypoxic MIL is in the environment of about 1% to about 3% oxygen gas. In some embodiments, the hypoxic MIL is in the environment of about 1% to about 4% oxygen gas. A hypoxic MIL is an MIL incubated for a period of time as described herein in a hypoxic environment such as that described herein. As described herein, a hypoxic MIL may also be activated in the presence of anti-CD3 / anti-CD28 beads or other similar activation reagents. Thus, a hypoxic MIL comprising CAR can also be an activated-hypoxic MIL.

VII. Treatment method

In some embodiments, cells expressing CAR (e. G., MIL) are provided. The cell (or parent cell) may be transfected with a vector comprising a nucleotide sequence encoding CAR. The vector may be a lentiviral vector (LV). For example, LV encodes CAR in combination with the intracellular domain of CD3ζ, CD28, 4-1BB, or any combination thereof and the antigen recognition domain of a specific antibody. Thus, in some cases, transduced MILs can induce CAR-mediated T-cell responses.

The use of CAR to convert the specificity of the primary MIL to tumor antigens is provided herein. Thus, in some embodiments, the step of administering to the subject an MIL expressing CAR, wherein CAR comprises a binding moiety that specifically interacts with a given target, e. G., An intracellular domain of human CD3ζ a < / RTI > chain portion, and a < RTI ID = 0.0 > co-stimulated < / RTI > signal transduction region.

In some embodiments, cell therapy is provided wherein the MIL is genetically modified to express CAR and the CAR-MIL is injected into a recipient in need thereof. The injected cells can kill the recipient's tumor cells (or other target). Unlike antibody therapies, CAR-MIL can be replicated in vivo, maintaining long-term persistence that can lead to continuous tumor control.

In some embodiments, the CAR-MIL may undergo a robust in vivo MIL expansion and may last for an extended period of time.

Cancers that can be treated include tumors that have not been vascularized or have not yet become substantially vascularized as well as vascularized tumors. Cancer can include non-solid tumors (e. G., Blood tumors, e. G., Leukemia and lymphoma) or can include solid tumors. Types of cancer to be treated with CAR include, but are not limited to, carcinoma, blastoma and sarcoma and certain leukemia or lymphoma malignancies, benign and malignant tumors and malignant tumors such as sarcoma, carcinoma and melanoma. Adult tumor / cancer and pediatric tumor / cancer are also included.

Blood cancers are blood or bone marrow cancers. Examples of blood (or blood) cancers include, but are not limited to, acute leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloproliferative cells, bone marrow cells, bone marrow mononuclear cells, mononuclear leukemia, Lymphocytes, Hodgkin's disease, non-Hodgkin's lymphoma (neutropenic and non-Hodgkin's lymphoma), multiple myeloma, valdendroma macroglobulinemia, small-cell lung cancer (Hodgkin's lymphoma), chronic myelogenous leukemia and chronic lymphocytic leukemia , Myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

Solid tumors are typically abnormal tissue masses that do not contain cysts or liquid areas. Solid tumors may be benign or malignant. Other types of solid tumors are termed types of cells that make them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovial sarcoma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphocytic malignancy, pancreatic cancer , Breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, watery thyroid carcinoma, papillary thyroid carcinoma, chromophilic adenocarcinoma, sebaceous adenocarcinoma, papillary carcinoma, papillary adenocarcinoma (E.g., brain stem (e. G., A glioma, e. G., A glioma, e. G., A glioma, e. G., A glioma), e. G. Glioma and mixed glioma), glioblastoma (also known as polymorphic glioblastoma), astrocytoma, CNS lymphoma, seed cell tumor, sublingual blastoma, schwannoma And a bell, noesilmak species, pineal species, vascular blastoma, jipjong auditory neurons, oligodendrocyte cell tumors, meningioma (menangioma), neuroblastoma, retinoblastoma, and brain metastasis).

In some embodiments, the antigen-binding moiety portion of CAR is designed to treat certain cancers. For example, CARs designed to target C19 include, but are not limited to, pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, salvage after homologous bone marrow transplantation, ≪ RTI ID = 0.0 > and the like. ≪ / RTI >

In some embodiments, CAR can be designed to target CD22 to treat diffuse large B-cell lymphoma.

In some embodiments, the cancer and disease include, but are not limited to, pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, allogeneic bone marrow transplantation post- CD19, CD20, CD22, and a combination of CARs targeting ROR1.

In some embodiments, the CAR can be designed to target mesothelin to treat mesothelioma, pancreatic cancer, ovarian cancer, and the like.

In some embodiments, the CAR can be designed to target CD33 / IL3Ra to treat acute myelogenous leukemia and the like.

In some embodiments, CAR can be designed to target c-Met to treat triple negative breast cancer, non-small cell lung cancer, and the like.

In some embodiments, the CAR can be designed to target PSMA to treat prostate cancer and the like.

In some embodiments, the CAR can be designed to target glycolipid F77 to treat prostate cancer and the like.

In some embodiments, the CAR can be designed to target EGFRvIII to treat gliobastoma and the like.

In some embodiments, CAR can be designed to target GD-2 to treat neuroblastoma, melanoma, and the like.

In some embodiments, the CAR can be designed to target the NY-ESO-1 TCR to treat myeloma, sarcoma, melanoma, and the like.

In some embodiments, the CAR can be designed to target the MAGE A3 TCR to treat myeloma, sarcoma, melanoma, and the like.

However, embodiments should not be construed as limited to the antigenic targets and diseases described herein. Rather, the embodiments should be construed to include any antigenic target associated with a disease in which CAR can be used to treat the disease.

CAR-modified MILs may also serve as a type of vaccine for in vitro immunization and / or in vitro therapy in a subject such as a human.

With respect to in vitro immunization, at least one of the following occurs in vitro before administration of the cell into the mammal: i) expansion of the cell, ii) introduction of the nucleic acid encoding the CAR into the cell and / or iii) Cryopreservation. In some embodiments, all steps are performed prior to administering the cells into the mammal.

The in vitro procedures are well known in the art and are discussed more fully below. Briefly, the cells are isolated from a mammal (e.g., a human) and genetically transformed (i. E., Transduced or transfected in vitro) into a vector expressing the CARs described herein. CAR-MIL may be administered to a mammalian recipient to provide therapeutic benefit. The mammalian recipient may be a human, and the CAR-MIL may be self associated with the recipient. Alternatively, the cells may be allogeneic, syngeneic or heterologous in connection with the recipient.

In addition to using a cell-based vaccine for in vitro immunization, compositions and methods for in vivo immunization to induce an antigen-induced immune response in a patient are provided herein.

Generally, activated and expanded cells, as described herein, can be used to treat and prevent diseases that occur in immunocompromised individuals. In some embodiments, a CAR-modified MIL is used in the treatment of CCL. In some embodiments, the cells are used in the treatment of patients at risk of developing CCL. Accordingly, there is provided a method of treating or preventing CCL, comprising administering a therapeutically effective amount of a CAR-modified MIL to a subject in need thereof.

The CAR-modified MIL may be administered alone or as a pharmaceutical composition in combination with a diluent and / or other ingredients such as IL-2 or other cytokines or cell populations. Briefly, a pharmaceutical composition may comprise a population of target cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions include buffers such as neutral buffered saline, phosphate buffered saline and the like; Carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; Polypeptides or amino acids such as glycine; Antioxidants; Chelating agents such as EDTA or glutathione; Adjuvants (e. G., Aluminum hydroxide); And preservatives. In some embodiments, the composition is formulated for intravenous administration.

The pharmaceutical composition may be administered in a manner suitable for the disease to be treated (or prevented). Appropriate dosages can be determined by clinical trials, although the dose and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient ' s disease.

When an "immunologically effective amount "," anti-tumor effective amount ","tumor- inhibiting effective amount ",or" therapeutically effective amount ", the precise amount of composition to be administered will depend on the age, weight, And the individual differences of the states. A pharmaceutical composition comprising an MIL as described herein may be administered at a dosage of 10 ⁴ to 10 ⁹ cells / kg body weight, preferably 10 ⁵ to 10 ⁶ cells / kg body weight, including all integer values within these ranges It can be generally stated that there is. The MIL composition may also be administered multiple times at such doses. Cells may be administered using immunization techniques generally known in the art (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). Optimal dosages and treatment regimes for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

Administration of the composition may be carried out in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intramuscularly, intravenously (iv) or intraperitoneally. In some embodiments, the MIL composition is administered to the patient by intradermal or subcutaneous injection. In some embodiments, the MIL composition is administered by intravenous injection. The composition of the MIL can be injected directly into, for example, a tumor, a lymph node, or an infection site.

In some embodiments, cells activated and enriched using the methods described herein or other methods known in the art in which the MILs are extended to therapeutic levels include, but are not limited to, agents such as antiviral therapy, cidofovir ) And interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or other treatment for patients with psoriasis or ephelizumab treatment for psoriasis (E. G., Before, concurrently or after) with the relevant treatment modality. In some embodiments, the MIL is selected from the group consisting of chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunosuppressants such as CAM PATH, anti- Can be used in combination with toxin, fluulbina, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and irradiation. These drugs inhibit calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase, which is crucial for growth factor-induced signal transduction (Liam et al., Liu et al., Cell 66: 807-815, 1991; Henderson et al., Immun 73: 316-321,1991; Bierer et al., Curr. Opin. Immun 5: 763-773,1993). In some embodiments, the cellular composition is administered in combination with bone marrow transplantation, chemotherapeutic agents such as MIL ablation therapy using fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH Before, concurrently with, or after) the patient. In some embodiments, the cell composition is administered after B-cell resection therapy, such as an agent that reacts with CD20, e.g., Rituxan. For example, in some embodiments, the subject is treated with high-dose chemotherapy followed by standard treatment with peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells described herein. In some embodiments, the expanded cells are administered before or after surgery.

The dosage of the therapeutic agent to be administered to the patient will depend on the exact nature of the condition being treated and the recipient of the treatment. Adjustment of the dosage for human administration can be carried out according to practices recognized in the art. For example, the capacity of CAMPATH will generally be in the range of 1 to about 100 mg for adult patients, usually daily for a period of 1 to 30 days. In some embodiments, the daily dose is between 1 and 10 mg / day, but in some cases, greater doses of up to 40 mg / day may be used (as described in U.S. Patent No. 6,120,766).

VIII. Object

The subject may be any organism, including MIL. For example, the subject may be selected from rodents, dogs, cats, pigs, sheep, cows, horses and primates. The subject may be a human or a mouse.

An object can have a neoplasm. The subject may be a benign neoplasm, a malignant neoplasm, or a secondary neoplasm. The neoplasm can be cancer. The neoplasm may be lymphoma or leukemia, such as chronic lymphocytic leukemia ("CLL") or acute lymphoblastic leukemia ("ALL"). The subject may have glioblastoma, hematoblastoma, breast cancer, head and neck cancer, kidney cancer, ovarian cancer, Kaposi sarcoma, acute myelogenous leukemia and B-line malignancy. The subject may have multiple myeloma.

The subject may be selected from the group consisting of acute myelogenous leukemia, adenocarcinoma, osteosarcoma, lymphoid leukemia, lymphoma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, B-lineage lymphoma, breast cancer, ovarian cancer, cervical cancer, A neoplasms of the glioma, a neoplasm of the glioma, a Hodgkin's lymphoma, a painless B-cell lymphoma, a leukemia, a lymphoma, a lung cancer, an extracellular lymphoma, a hematoblastoma, a melanoma, a neuroblastoma, a prostate cancer, a vesicular lymphoma, a kidney cell carcinoma, .

Example

The following examples are illustrative of the methods and compositions described herein, but are not limited thereto. Other suitable variations and modifications of various conditions and parameters which will be apparent to those skilled in the art and which are generally encountered in therapy are within the spirit and scope of the specific embodiments.

Example  1: MIL-CAR is used for the treatment of B-cell lymphoma.

MIL was obtained from a subject with B-cell lymphoma. Briefly, after obtaining a bone marrow sample from a subject, the cells were incubated under hypoxic conditions in the presence of anti-CD3 / anti-CD28 beads and cytokines as described in WO2016037054, incorporated herein by reference. Nucleic acid molecules encoding CAR, including the extracellular domain of CD19, the transmembrane domain of CD19, and the intracellular domain of CD3ζ and 4-1BB, were transfected into MIL. The cells were then grown and expanded under normoxic conditions. Activated and expanded MIL was administered to subjects with B-cell lymphoma. B-cell lymphoma of the subject showed a difference. In summary, the embodiments and examples provided herein demonstrate that cells expressing CAR can be effectively used to treat cancer.

Example  2: MIL-CAR is used to treat multiple myeloma.

MIL was obtained from a subject with multiple myeloma. Briefly, after obtaining a bone marrow sample from a subject, the cells were incubated under hypoxic conditions in the presence of anti-CD3 / anti-CD28 beads and cytokines as described in WO2016037054, incorporated herein by reference. Nucleic acid molecules encoding CAR were transfected into MIL, including the extracellular domain of CD38, the transmembrane domain of CD8, and the intracellular domain of CD3ζ and 4-1BB. The cells were then grown and expanded under normoxic conditions. Activated and expanded MIL was administered to subjects with multiple myeloma. The multiple myeloma of the subject showed a difference.

Non-patent publications, including any US patents, US patent application disclosures, US patent applications, foreign patents, foreign patent applications and CAS numbers mentioned herein and / or listed in the application datasheet, Lt; / RTI >

Claims (35)

As a cell comprising a chimeric antigen receptor ("CAR"),
The cell is a bone marrow infiltrating lymphocyte ("MIL");
CAR comprises an extracellular domain capable of binding to a ligand;
CAR is a cell that contains an intracellular domain that can initiate an intracellular signaling cascade. 2. The cell of claim 1, wherein the cell is CD3 ⁺ . 3. The cell according to claim 1 or 2, wherein the cell is CD4 ⁺ . 4. The cell according to any one of claims 1 to 3, wherein the cell is CD8 ⁺ . 5. The cell according to any one of claims 1 to 4, wherein the cell is CD45RO +. 6. The cell according to any one of claims 1 to 5, wherein the cell is CD62L +. 7. The cell according to any one of claims 1 to 6, wherein the cell is CXCR4 +. 8. The cell according to any one of claims 1 to 7, wherein the cell is 4-1BB ⁺ . Any one of claims 1 to 8 according to any one of, wherein the interferon-γ ⁺ cells are the cells. 10. The cell according to any one of claims 1 to 9, wherein the cell is CD138 ⁺ . 11. The cell according to any one of claims 1 to 10, wherein the cell is CD33 ⁺ . 12. The cell according to any one of claims 1 to 11, wherein the cell is CD34 ^- . 13. The cell according to any one of claims 1 to 12, wherein the ligand is a molecule expressed in a neoplastic cell. 14. The method of claim 13, wherein the ligand is selected from the group consisting of glioma-associated antigen, cancer embryonic antigen (CEA), beta -human chorionic gonadotropin, alpha-fetoprotein 1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mutant hsp70-Ifetoprotein, lectin-reactive AFP, thyroglobulin, RAGE- 2, M-CSF, prostase, prostate-specific antigen (PSA), prostate acid phosphatase (PAP), NY-ESO-1, LAGE-1a, p53, , PSMA, Her2 / neu, survivin, telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, IGF-I receptor, mesothelin, MART-1, tyrosinase, GP 100, HER-2 / Neu / ErbB- 2, CD19, CD20, CD37, MART-1 / MelanA ("MART-I"), gp100 1, GAGE-2, p15, p53, Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK , MYL-RAR, EBVA, E6, E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erBB- -72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72 Alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 \ CA 27.29 \ BCAA, CA 195, CA 242, CA- 50, CAM 43, CD 68 \ MAG-2 binding, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, Protein Cyclophilin C-related protein, TAAL6, TAG72, TLP, or TPS. 14. The method of claim 13, wherein the ligand is selected from the group consisting of CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY- ESO- cell. 14. The method of claim 13, wherein the ligand is selected from the group consisting of alpha -folate receptor, carbonic anhydrase 9 ("CAIX"), CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7, CD44v7, (EGF-2), epithelial glycoprotein 40 ("EGF-40"), receptor tyrosine-protein kinase erbB-2 (HER2; Neu; CD340), receptor (HER4), folate-binding protein ("FBP"), fetal acetylcholine receptor, GD2, GD3, interleukin-13 receptor subunit alpha (&Quot; KDR "), kappa light chain, Lewis Yantigen ("LeY"), L1 cell adhesion molecule, MAGE-A1, mesothelin Cell, a mucin 1 (cell surface) ("MUC1"), a prostate stem cell antigen, a prostate-specific membrane antigen, a tumor-associated glycoprotein 72 ("TAG-72"), or VEGF-R2. 17. The cell according to any one of claims 1 to 16, wherein the ligand is a molecule expressed by a pathogen. 18. The cell of claim 17, wherein the pathogen is a virus, a bacterium, a fungus, a parasite, or a viroid. 19. A cell according to any one of claims 1 to 18, wherein the extracellular domain of CAR comprises a single-chain variable fragment ("scFv") domain. 20. The cell of any one of claims 1 to 19, wherein the intracellular domain of CAR comprises the intracellular signaling domain of CD3ζ, 4-1BB, and / or CD28. 20. A method for treating a condition in a subject, comprising administering to the subject a cell of any one of claims 1 to 20. 22. The method of claim 21, wherein the method comprises administering to the subject a plurality of cells according to any one of claims 1 to 20. A method of producing a recombinant MIL,
Obtaining a bone marrow comprising MIL; And
Transforming or transducing MIL with a nucleic acid encoding a chimeric antigen receptor. 24. The method of claim 23, wherein bone marrow is obtained from a subject. 25. The method of claim 24, wherein the subject has a neoplasm. 25. The method of claim 24, wherein the subject has an autoimmune disease. 25. The method of claim 24, wherein the subject has an infection caused by a pathogen. 28. The method according to any one of claims 23 to 27, further comprising activating the MIL. 29. The method according to any one of claims 23 to 28, further comprising extending the MIL. 30. The method according to any one of claims 23 to 29, wherein the method comprises producing a plurality of recombinant MILs. 24. The method of claim 23, further comprising incubating the MIL under hypoxic conditions before transfection, transformation or transfection of the MIL with a nucleic acid encoding a chimeric antigen receptor. 32. The method of claim 31, wherein the hypoxic condition comprises from about 0.5% to about 5% oxygen gas. 32. The method of claim 31, wherein the hypoxic condition comprises from about 1% to about 2% oxygen gas. 32. The method of claim 31, further comprising the step of transfecting, transfecting or transfecting MIL with a nucleic acid encoding a chimeric antigen receptor and then incubating the MIL under normal oxygen conditions. 32. The method of claim 31, further comprising contacting the MIL with an anti-CD3 / anti-CD28 bead while incubating the MILS under hypoxic conditions.